Tobacco-related deaths cost the United States approximately $200 billion each year. Nicotine is singularly responsible for the dependence-forming properties of tobacco smoking and, once introduced to the body, affects the brain within 15 seconds. Breaking nicotine addiction is challenging and relapse rates remain high. The extremely rapid timescale of nicotine action renders existing techniques for studying nicotine accumulation in the brain (e.g., microdialysis, PET, radioimmunoassay) ill-suited for addiction studies in freely moving animals. Biosensors are a proven technology for monitoring real-time changes in CNS neurochemical concentrations on a second-by-second basis. The overall goal of this proposal is the delivery of a biosensor that can detect nicotine in a freely moving animal at relevant in vivo concentrations and that is compatible with Pinnacle's existing turn-key systems. The most critical component of a biosensor is the enzyme used as the biorecognition element, and no aspect of a biosensor's final design is as vital as a properly folded enzyme with sufficient activity and stability profiles. In Phase I, we were highly successful in providing proo-of-concept for two critical components of the nicotine biosensor: (1) conversion of 6-hydroxynicotine oxidase into an oxidase with an enhanced kcat for nicotine (~0.2 sec-1) while maintaining a Km that is 1000-fold above expected in vivo concentrations, and (2) development of new, thinner membranes to support nicotine monitoring. We also successfully implemented a random selection and screening strategy that gave rise to new mutations that enhanced the performance characteristics of the engineered nicotine oxidase enzyme. Phase II will complete the nicotine enzyme optimization through ongoing directed evolution of the nicotine oxidase enzyme. We will also continue the ongoing optimization of new films to improve biosensor sensitivity. The completion of this proposal will provide two important innovations to the scientifc community: 1) A nicotine biosensor suitable for addiction studies that provides second-by-second changes of nicotine concentration in the CNS. 2) A refined approach for the development of new biosensors that target analytes important for addiction and for which no oxidase enzyme currently exists (i.e., cocaine and caffeine). Pinnacle will work in conjunction with an interdisciplinary consortium of leading scientists at the University of Kansas. When completed, this will be the first commercial biosensor specifically designed to record nicotine in rodents and will represent the first commercialized biosensor for in vivo use that is based on an engineered oxidase enzyme for an analyte for which no enzyme previously existed.